Detection and Elimination of An Antigenic Non-Human Sialic Acid in Human Embryonic Stem Cells
This application addresses a “significant technical hurdle that needs to be overcome before stem cell research can be effectively translated to the clinic” and falls in the category of “discovery and evaluation of novel tools and technologies”. Proposed work will be performed by researchers at a California-based company and [REDACTED] with the aim of developing diagnostics, tools and possible therapies involving human embryonic stem cells (hESc).
The surface of hESc is covered with a dense, complex array of sugar chains. The focus of our work is on a non-human cell surface sugar molecule called N-glycolylneuraminic Acid (Neu5Gc), which is naturally present in large amounts on the cell surfaces and secreted glycoproteins of most animals but not humans. Humans have a genetic mutation that prevents Neu5Gc production, and instead accumulate the precursor form of the molecule N-acetylneuraminic Acid (Neu5Ac). However, human cells can incorporate Neu5Gcfrom external sources. Standard hESc culture methods use animal-derived products and mouse fibroblast feeder layers containing Neu5Gc, which is thus taken up, metabolically processed and expressed on the hESc cell surface-as if it were produced naturally within these cells. The hESc remain contaminated by Neu5Gc even when grown in serum-free culture conditions because most serum replacements also contain animal-derived products. This Neu5Gc contamination of hESc is of significance because all humans havecirculating anti-Neu5Gc antibodies that can initiate in an inflammatory response against this foreign molecule. Therefore, any hESc generated using currently standard conditions would result in Neu5Gc-contaminated grafts, which, if placed into human patients, would be at risk of reaction, or even outright rejection. In light of this very practical concern, the overall goals of the proposed research is to:
1.Show that Neu5Gc-deficient mouse embryonic fibroblasts can function as a feeder layer for growth and maintenance of hESc. The use of these fibroblasts could reduce the contamination and help minimize the risk of rejection. 2. Use a newly developed metabolic technique to eliminate and/or prevent incorporation of Neu5Gc into hESc. 3.Optimize antibody and other techniques for rapid and sensitive detection of Neu5Gc in the quality assurance process of hESc, and hESc-derived biotherapeutics.
The technical approaches proposed are: 1.Optimize the use of Neu5Gc-free mouse fibroblasts as feeder layers in human embryonic stem cell cultures. 2.Develop and optimize approaches to eliminate Neu5Gc or prevent incorporation in the first place. 3.Evaluate and optimize use of antibodies and other techniques for sensitive and specific detection of Neu5Gc-containing epitopes.
In summary, it is highly likely that safe, effective, unique solutions to Neu5Gc contamination in hESc research will be offered by a California-based enterprise to research and industrial scientists – and eventually to patients.
Currently, the State of California is unique because it is the only state that uses bond proceeds for its Institute for Regenerative Medicine. The tools and technologies grant awards are meant to provide standardized processes that will overcome current roadblocks, which will in turn assure clinically useful quantities of uncontaminated stem cells in sufficient quantities for the production of stem cell-derived biotherapeutic products. The roadblock that this CIRM Tools and Technologies proposal addressesis the elimination of a non-human contaminant from human stem cells cultured under conventional methods. This is an important innovation that will give the state of California the ability to stay at the forefront of this emerging biomedical market.
The expected outcome from the funding of our proposed work would benefit California in many ways. All proprietary technologies and methods used to remove this specific type of hESc contamination will have been discovered or developed in California universities or small California biotechnology companies. The funding of this proposal would also allow California to promote interactions between basic academic researchers and scientists in industry who translate recent discoveries into financially beneficial iomedical products. This by itself would bring esteem to the state as being supportive in innovative technologies necessary at the very basic level of hESc maintenance. In addition, the State of California would continue to benefit financially and biomedically because all the entities named here would remain in California and strive to use vendors and purchase services found in California to keep grant monies in the state.
Funding the proposed work for the company,[REDACTED] will also show California as a forward-looking state because it is a woman-owned small business. The already funded principal investigator, [REDACTED], is a California-born Latina, a product of the California education system, and as such would contribute to increasing California-educated California scientists. In short, funding [REDACTED] would increase the number of under-represented minorities in business and graduate-level biomedical scientists in Clifornia.
If funded, the proposed work could eliminate a non-human contaminant hESc that has been shown to be harmful because all humans produce antibodies against it and are known to participate in inflammation and possibly the rejection of tissue transplants. California would be unique because it would be the only state in the U.S. to offer the technology to detect Neu5Gc contamination and quickly remove an immunologically significant roadblock for the large-scale production of clinically relevant uncontaminated ESc. We feel that the development of this technology that we have shown to work in on a small scale will allow California to successfully compete, at a time when the U.S. is losing itís competitive edge in the world of stem cell research.